Eph receptors are the largest family of receptor tyrosine kinases and play key roles in cell targeting All of the Eph receptors possess a SAM domain, a roughly 70 amino acid protein module. The SAM domain has been found in over 80 proteins playing important roles in signal transduction, development and other regulatory processes. Work in a number of laboratories, including my own, demonstrates that SAM domains can form specific homo- and heterodimers. As part of an effort to understand the mechanism of Eph receptor activation, we propose to investigate the rules that specify SAM domain interactions. Because of the wide distribution of the SAM domain, our work will have implications in many areas of signal transduction. The specific aims are: 1. Determine the structure of the SAM domain from Eph receptor EphB2 (EphB2-SAM) by x-ray crystallography. We have already obtained high quality crystals of the EphB2-SAM domain. The structure will play an important role in understanding the functions of these domains, providing a rational basis for mutagenesis experiments and possibly leading to new ideas about function. II. Identify key determinants of SAM domain heterodimerization in a simple system. To learn the rules that govern SAM domain association it is essential to identify which residues are critical for binding. Because SAM domain interactions in Eph receptors appear to be complex and regulated, we plan to explore the mechanism of SAM domain association using a simpler system. We have shown that the SAM domains of the yeast S. pombe proteins Byr2 and Ste4 form a heterodimer, and propose to identify the key residues by characterizing mutant proteins that can no longer form heterodimers. III. Investigate Eph receptor SAM domain binding. Results from my laboratory indicate that the Eph receptor SAM domains can Oligomerize, but that the association is regulated by phosphorylation. We plan to characterize SAM domain mediated oligomerization in the Eph receptors, identify residues important for the interaction and examine the biological consequences of mutants that do not oligomerize.